The peripheral auditory system communicates with the central nervous system through synapses between the sensory cells and the spiral ganglion neurons. This single synapse carries information regarding frequency, intensity and timing that is used by the CNS for to extract all information provided by the auditory modality. The synapse is specialized to operate across a broad frequency range, to respond to graded stimuli in a linear manner. The synapse maintains high rates of release and does not fatigue and is sensitive to receptor potentials often below 1 mV. The goal of this proposal is to delineate the mechanisms underlying these unusual synaptic properties. Both pre and postsynaptic mechanisms will be investigated. Specific Aim 1 addresses presynaptic issues and has three subaims; to identify the Ca2+ dependent components of vesicle release and trafficking, to determine the kinetics of release while identifying the rate limiting steps of trafficking and release, and to elucidate tonotopic differences in release properties, characterizing Ca2+ homeostatic mechanisms that regulate release and identifying phosphorylation dependent regulation of release and trafficking. Specific Aim 2 investigates postsynaptic processing with three goals; first to determine if there are tonotopic variations in afferent electrical properties that might serve to enhance frequency selectivity such as the kinetics and magnitude of voltage-dependent processes, second to characterize synaptic transmission at different locations to identify differences in receptor types and numbers and third to investigate mechanisms associated with multivesicular release. Structural, immunocytochemical, optical, electrophysiological and theoretical tools including several new technologies are included to better enable us to investigate these issues. Given that any deficits in synaptic transmission at the hair cell afferent fiber synapse results in auditory deficits, identifying novel mechanisms may provide unique avenues for intervention. PUBLIC HEALTH RELEVANCE All of the signal processing that happens at the auditory periphery is communicated to the central nervous system through the hair cell afferent fiber synapse. Any action that results in a loss of fidelity of this synapse leads to hearing loss and deafness. The requirements of this synapse are quite demanding and result in several unique specializations including: a graded and linear response to stimulus, an amazing lack of fatigue in the face of continual and often high release rates and postsynaptic firing rates, and the ability to phase-lock across several orders of magnitude of frequencies. Mechanisms underlying these specialized properties are unknown but should offer sites for intervention and prevention of noise induced and even ototoxic induced hearing loss. The primary goal of this work is to identify and characterize these mechanisms.